The present invention relates to the treatment of hepatic cirrhosis and, in particular, to the treatment of hepatic cirrhosis with quinazolinone derivatives such as Halofuginone.
Hepatic cirrhosis has a number of causes, including hepatic fibrosis caused by chronic alcoholism, malnutrition, hemochromatosis, passive congestion, hypercholesterolemia, exposure to hepatotoxic chemical substances, exposure to drugs, immune reactions, genetically determined sensitivities to certain substances as seen with copper in Wilson""s disease and infections such as viral hepatitis, syphilis and various parasitic infections including, but not limited to, Schistosomiasis mansoni and S. japonica. For reasons given in greater detail below, the disease is currently incurable and frequently fatal.
The pathogenesis of hepatic cirrhosis progresses in a number of stages. First, an enlarged liver is seen with various fatty changes. Next, overt fibrosis is evident with a concomitant decrease in liver function. Finally, atrophy of the liver begins, with a corresponding reduction in the size and functionality of the liver. Necrosis of the liver can be seen at any stage, but is particularly pronounced by late stage cirrhosis. Microscopically, a complete disruption of the normal architecture of the liver is evident.
Outside of the liver, other pathological changes become evident as cirrhosis progresses. Portal circulation is reduced as fibrotic tissue is formed in the liver, further reducing liver functionality. This reduced circulation causes an increase in collateral venous circulation, particularly in the esophagus. These esophageal blood vessels can rupture, causing fatal hemorrhage. Thus, cirrhosis is an entire pathological process with effects that are not limited to the liver, although the root causes can be found in specific pathological changes to the liver itself.
One necessary step in the pathogenesis of hepatic cirrhosis is the formation of fibrotic tissue in the liver. Hepatic fibrosis is a feature of most chronic liver diseases, not just cirrhosis [S. L. Friedman, New Eng. J. Med., 328:1828-35, 1993]. In hepatic fibrosis, connective tissue accumulates in the liver, replacing normal hepatic parenchymal tissue, and reducing liver functionality. The fibrotic tissue replaces more complex normal liver tissue in a pathological process which reduces the amount of liver tissue available for normal functions, such as the removal of toxic substances from the blood, and which progressively disrupts intrahepatic blood flow. The formation of fibrotic tissue in the liver is characterized by the deposition of abnormally large amounts of extracellular matrix components, including at least five types of collagen, in particular collagen types I, III, and IV, as well as other matrix proteins [L. Ala-Kokko, Biochem. J, 244:75-9, 1987].
The synthesis of collagen is also involved in a number of other pathological conditions. For example, clinical conditions and disorders associated with primary or secondary fibrosis, such as systemic sclerosis, graft-versus-host disease (GVHD), lung fibrosis and a large variety of autoimmune disorders, are distinguished by excessive production of connective tissue, which results in the destruction of normal tissue architecture and function. These diseases can best be interpreted in terms of perturbations in cellular functions, a major manifestation of which is excessive collagen synthesis and deposition. The crucial role of collagen in fibrosis has prompted attempts to develop drugs that inhibit its accumulation [K. I. Kivirikko, Annals of Medicine, Vol. 25, pp. 113-126 (1993)].
Such drugs can act by modulating the synthesis of the procollagen polypeptide chains, or by inhibiting specific post-translational events, which will lead either to reduced formation of extra-cellular collagen fibers or to an accumulation of fibers with altered properties. Unfortunately, only a few inhibitors of collagen synthesis are available, despite the importance of this protein in sustaining tissue integrity and its involvement in various disorders.
For example, cytotoxic drugs have been used in an attempt to slow the proliferation of collagen-producing fibroblasts [J. A. Casas, et al., Ann. Rhem. Dis., 46: 763, 1987], such as colchicine, which slows collagen secretion into the extracellular matrix [D. Kershenobich, et al., N. Engl. J. Med., 318:1709, 1988], as well as inhibitors of key collagen metabolism enzymes [K. Karvonen, et al., J. Biol Chem., 265: 8414, 1990; C. J. Cunliffe, et al., J. Med. Chem., 35:2652, 1992].
Unfortunately, none of these inhibitors are collagen-type specific. Also, there are serious concerns about the toxic consequences of interfering with biosynthesis of other vital collagenous molecules, such as Clq in the classical complement pathway, acetylcholine esterase of the neuro-muscular junction endplate, conglutinin and liver surfactant apoprotein.
Other drugs which can inhibit collagen synthesis, such as nifedipine and phenytoin, inhibit synthesis of other proteins as well, thereby non-specifically blocking the collagen biosynthetic pathway [T. Salo, et al., J. Oral Pathol. Med., 19: 404,1990].
Collagen cross-linking inhibitors, such as xcex2-amino- propionitrile, are also non-specific, although they can serve as useful anti-fibrotic agents. Their prolonged use causes lathritic syndrome and interferes with elastogenesis, since elastin, another fibrous connective tissue protein, is also cross-linked. In addition, the collagen cross-linking inhibitory effect is secondary, and collagen overproduction has to precede its degradation by collagenase. Thus, a type-specific inhibitor of the synthesis of collagen itself is clearly required as an anti-fibrotic agent.
Such a type-specific collagen synthesis inhibitor is disclosed in U.S. Pat. No. 5,449,678 for the treatment of a fibrotic condition. This specific inhibitor is a composition with a pharmaceutically effective amount of a pharmaceutically active compound of a formula: 
wherein:
R1 is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy; R2 is a member of the group consisting of hydroxy, acetoxy and lower alkoxy; and R3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl; wherein n is 1 or 2. Pharmaceutically acceptable salts thereof are also included. Of this group of compounds, Halofuginone has been found to be particularly effective for such treatment.
U.S. Pat. No. 5,449,678 discloses that these compounds are effective in the treatment of fibrotic conditions such as scleroderma and GVHD. PCT Application No. WO 96/06616 further discloses that these compounds are effective in treating restenosis. The two former conditions are associated with excessive collagen deposition, which can be inhibited by Halofuginone. Restenosis is characterized by smooth muscle cell proliferation and extracellular matrix accumulation within the lumen of affected blood vessels in response to a vascular injury [Choi et al., Arch. Surg., 130:257-261, 1995]. One hallmark of such smooth muscle cell proliferation is a phenotypic alteration, from the normal contractile phenotype to a synthetic one. Type I collagen has been shown to support such a phenotypic alteration, which can be blocked by Halofuginone [Choi et al., Arch. Surg., 130: 257-261, 1995; U.S. Pat. No. 5,449,678].
However, the in vitro action of Halofuginone does not always predict its in vivo effects. For example, Halofuginone inhibits the synthesis of collagen type I in bone chrondrocytes in vitro, as demonstrated in U.S. Pat. No. 5,449,678. However, chickens treated with Halofuginone were not reported to have an increased rate of bone breakage, indicating that the effect is not seen in vivo. Thus, the exact behavior of Halofuginone in vivo cannot always be accurately predicted from in vitro studies.
Furthermore, the ability of Halofuginone or other related quinazolinone to block or inhibit pathological processes related to hepatic cirrhosis has not been demonstrated. Other inhibitors of collagen synthesis, cross-linking and deposition, such as corticosteroids, penicillamine, methotrexate and colchicine, have been tested for their therapeutic effect on hepatic fibrosis, but have not proved effective [S. L. Friedman, New Eng. J. Med., 328:1828-35, 1993]. Although Halofuginone has been shown to have a specific inhibitory effect on the synthesis of type I collagen, such inhibition has not been otherwise shown to be efficacious in the treatment of hepatic cirrhosis. Indeed, hepatic cirrhosis has a high mortality rate, as currently available therapeutic options have significant side effects and are not generally efficacious in slowing or halting the progression of the fibrosis. Furthermore, many other types of extracellular matrix components are deposited during the pathogenesis of hepatic fibrosis, including at least five types of collagen, in particular collagen types I, III, and IV, as well as other matrix proteins [L. Ala-Kokko, Biochem. J, 244:75-9, 1987]. Thus, merely inhibiting synthesis of collagen type I would not necessarily slow or halt the development of hepatic fibrosis.
Thus, simply administering known in vitro inhibitors of collagen synthesis, deposition and cross-linking in an attempt to treat hepatic cirrhosis is ineffective. Clearly, new treatments for this incurable disease are required which specifically slow or halt the pathogenesis of fibrosis, without non-specific or toxic side effects.
There is thus a widely recognized need for, and it would be highly advantageous to have, a treatment for liver cirrhosis and fibrosis which inhibits fibrogenesis substantially without undesirable non-specific or toxic side effects.
Unexpectedly, it has been found, as described in the examples below, that Halofuginone can also inhibit the pathophysiological process of hepatic fibrosis in vivo, possibly by inhibiting collagen type I synthesis, although another mechanism or mechanisms could also be responsible. While inhibition of collagen type I synthesis is proposed as a plausible mechanism, it is not desired to be limited to a single mechanism, nor is it necessary since the in vivo data presented below clearly demonstrate the efficacy of Halofuginone as an inhibitor of hepatic fibrosis in vivo.
According to the teachings of the present invention, there is provided a method for the treatment of hepatic cirrhosis in a subject, comprising the step of administering to the subject a pharmaceutically effective amount of a compound having a formula: 
wherein:
R1 is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy;
R2 is a member of the group consisting of hydroxy, acetoxy and lower alkoxy, and
R3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl;
wherein n is 1 or 2; and pharmaceutically acceptable salts thereof.
Preferably, the hepatic cirrhosis is caused by contact with a hepatotoxic chemical substance.
According to another embodiment of the present invention, there is provided a method for the treatment of hepatic fibrosis in a subject, the hepatic fibrosis being caused by contact with a hepatotoxic chemical substance, the method comprising the step of administering to the subject a pharmaceutically effective amount of a compound having a formula: 
wherein:
R1 is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy;
R2 is a member of the group consisting of hydroxy, acetoxy and lower alkoxy, and
R3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl;
wherein n is 1 or 2; and pharmaceutically acceptable salts thereof.
According to preferred embodiments of the present invention, the hepatic fibrosis is caused by a factor selected from the group consisting of chronic alcoholism, malnutrition, hemochromatosis, passive congestion, hypercholesterolemia, exposure to poisons or toxins, exposure to drugs, immune reactions, genetically determined sensitivities to a certain substance and infections.
More preferably, the hepatic fibrosis is caused by a factor selected from the group consisting of viral hepatitis, syphilis and a parasitic infection.
Most preferably, said parasitic infection is selected from the group consisting of Schistosomiasis mansoni and S. japonica. 
According to yet another embodiment of the present invention, there is provided a method for the treatment of an existing condition of hepatic cirrhosis in a subject, comprising the step of administering to the subject a pharmaceutically effective amount of a compound having a formula: 
wherein:
R1 is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy;
R2 is a member of the group consisting of hydroxy, acetoxy and lower alkoxy, and
R3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl;
wherein n is 1 or 2; and pharmaceutically acceptable salts thereof.
According to further preferred embodiments of the present invention, the compound is preferably Halofuginone. Hereinafter, the term xe2x80x9cHalofuginonexe2x80x9d is defined as a compound having a formula: 
and pharmaceutically acceptable salts thereof. The composition preferably includes a pharmaceutically acceptable carrier for the compound.
According to another embodiment of the present invention, there is provided a method of manufacturing a medicament for treating hepatic cirrhosis, the hepatic cirrhosis being caused by contact with a hepatotoxic chemical substance, the method including the step of placing a pharmaceutically effective amount of a compound in a pharmaceutically acceptable carrier, the compound being a member of a group having a formula: 
wherein:
R1 is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R2 is a member of the group consisting of hydroxy, acetoxy, and lower alkoxy, and R3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl; wherein n is 1 or 2. Pharmaceutically acceptable salts thereof are also included.
According to yet another embodiment of the present invention, there is provided a composition for treating hepatic fibrosis, the hepatic fibrosis being caused by contact with a hepatotoxic chemical substance, the composition including a pharmaceutically effective amount of a compound in combination with a pharmaceutically acceptable carrier, the compound being a member of a group having a formula: 
wherein:
R1 is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R2 is a member of the group consisting of hydroxy, acetoxy, and lower alkoxy; and R3 is a member of the group consisting of hydrogen and lower alkenoxy; wherein n is 1 or 2. Pharmaceutically acceptable salts thereof are also included.
According to the present invention, there is also provided a method of manufacturing a medicament for treating hepatic fibrosis, the hepatic fibrosis being caused by contact with a hepatotoxic chemical substance, the method including the step of placing a pharmaceutically effective amount of a compound in a pharmaceutically acceptable carrier, the compound being a member of a group having a formula: 
wherein:
R1 is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R2 is a member of the group consisting of hydroxy, acetoxy, and lower alkoxy; and R3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl; wherein n is 1 or 2. Pharmaceutically acceptable salts thereof are also included.
According to still another embodiment of the present invention, there is provided a method for the treatment of hepatic cirrhosis in a subject, with the proviso that the hepatic cirrhosis is not caused by chronic alcoholism, viral hepatits or an autoimmune condition, the method comprising the step of administering to the subject a pharmaceutically effective amount of a compound having a formula: 
wherein:
R1 is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R2 is a member of the group consisting of hydroxy, acetoxy and lower alkoxy, and R3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl; wherein n is 1 or 2; and pharmaceutically acceptable salts thereof.
According to yet another embodiment of the present invention, there is provided a method for the treatment of hepatic fibrosis in a subject, with the proviso that the hepatic fibrosis is not caused by chronic alcoholism, viral hepatitis or an autoimmune condition, the method comprising the step of administering to the subject a pharmaceutically effective amount of a compound having a formula: 
wherein:
R1 is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R2 is a member of the group consisting of hydroxy, acetoxy and lower alkoxy, and R3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl; wherein n is 1 or 2; and pharmaceutically acceptable salts thereof.
Hereinafter, the term xe2x80x9csubjectxe2x80x9d refers to the human or lower animal to whom Halofuginone was administered. The term xe2x80x9cpatientxe2x80x9d refers to human subjects. The term xe2x80x9ctreatmentxe2x80x9d includes slowing or halting the progression of hepatic cirrhosis or fibrosis once it has arisen. The phrase xe2x80x9csubstantially preventing the genesisxe2x80x9d of hepatic cirrhosis or fibrosis is understood to refer to the prevention of the appearance of clinical or preclinical symptoms of these conditions, including the prevention of those symptoms which are indirectly related to the fibrotic and cirrhotic processes themselves, such as hemorrhage from esophageal blood vessels.
Hereinafter, the term xe2x80x9chepatotoxic chemical substancexe2x80x9d refers to chemicals which are not normally consumed or introduced into the circulation, and which have a harmful effect on the subject upon contact with the subject through introduction into the circulation of the subject, consumption by the subject or introduction to the subject through another route of administration.
Although the specific quinazolinone derivative xe2x80x9cHalofuginonexe2x80x9d is referred to throughout the specification, it is understood that other quinazolinone derivatives may be used in its place, these derivatives having the formula: 
wherein:
R1 is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy; R2 is a member of the group consisting of hydroxy, acetoxy and lower alkoxy, and R3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl; wherein n is 1 or 2. Pharmaceutically acceptable salts thereof are also included.
While the invention will now be described in connection with certain preferred embodiments in the following figures and examples so that aspects thereof may be more fully understood and appreciated, it is not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the scope of the invention as defined by the appended claims. Thus, the following figures and examples which include preferred embodiments will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of formulation procedures as well as of the principles and conceptual aspects of the invention.